The broad objective of this proposal is to understand the role of cytoskeletal proteins in establishing the unique cell shapes of neurons. Neurofilaments are a neuron-specific major component of the axonal cytoskeleton, but their function in axons is unknown. Knowing how neurofilaments and other cytoskeletal components help establish and maintain the axon is central to understanding both the capacity of the nervous system to recover after injury and the deterioration of axons in neural degenerative diseases. The nervous system of the frog, Xenopus laevis, has proven itself as a lower vertebrate model with high connectivity to other animals. Its rapidly developing embryo is easily accessible during early stages of neuronal differentiation, and macromolecules, such as antibodies, mRNAs and DNAs, can be injected into embryonic blastomeres to alter development of their descendent cells. Growing Xenopus axons contain the middle molecular weight neurofilament protein, NF-M; and antibodies to NF-M, injected into blastomeres, subsequently affect both the distribution of neurofilaments and axonal development, as observed with other antibodies that label axons. To clarify the relationship between the antibody effects and axon development, methods will be used to observe process outgrowth independently of expressed axonal markers. This will be done by labeling growing axons with the lypophilic dye, diI, and by observing affected neurons differentiating in culture. Electron microscopy will be used to observe effects on the number and arrangement of neurofilaments and microtubules in such neurons. Alternative reagents, such as Fab fragments from NF-M antibodies, and antisense methods that block NF-M translation, will also be used to confirm that the effects of injected NF-M antibodies relate directly to the function of NF-M. Fab fragments, together with peptides from the C-terminal domain of NF-M, will also test the possibility that specific domains of the NF-M molecule contribute to function by interacting with other components of the cytoskeleton. Lastly, mRNA (or plasmids designed to express NF-M mRNA) will be injected to see what happens to axonal development when NF-M is present too soon and at higher levels than normal. In addition to helping to answer why developing axons contain NF-M, this work will establish paradigms for using Xenopus to study other cytoskeletal proteins in neuronal differentiation.